Potyviruses are a distinct group of plant viruses which are pathogenic to various crops. Potyviruses include watermelon mosaic virus II (WMVII); papaya ringspot virus strains papaya ringspot and watermelon mosaic I(PRV-p and PRV-w), two closely related members of the plant potyvirus group which were at one time classified as distinct virus types, but are presently classified as different strains of the same virus; zucchini yellow mosaic virus (ZYMV); and many others. These viruses consist of flexous, filamentous particles of dimensions approximately 780.times.12 nanometers. The viral particles contain a single-stranded RNA genome containing about 10,000 nucleotides of positive (+, coding, or sense) polarity. Translation of the RNA genome of potyviruses shows that the RNA encodes a single large polyprotein of about 330 kD. The polyprotein contains several proteins, one of which is a 49 kD protease that is specific for the cleavage of the polyprotein into at least six (6) other peptides. One of the proteins contained within this polyprotein is a 35kD capsid or coat protein which coats and protects the viral RNA from degradation.
The genome organization of several viruses belonging to the potyvirus family group has been studied in detail, in particular tobacco etch virus, tobacco vein mottling virus and pepper mottle virus. In each case, the location of the coat protein gene has been at the 3'-end of the RNA, just prior to a stretch of 200 to 300 bases) terminal adenine nucleotides residues. The location of the 49 kD protease gene appears to be conserved in these viruses. In the tobacco etch virus, the protease cleavage site has been determined to be the dipeptide Gln-Ser, Gln-Gly or Gln-Ala. Conversation of these dipeptides as the cleavage sites in these viral polyproteins is apparent from the sequences of the above-listed potyviruses.
Expression of the coat protein genes from tobacco mosaic virus, alfalfa mosaic virus, cucumber mosaic virus, and potato virus X in transgenic plants has resulted in plants which are resistant to infection by the respective virus. In order to produce such transgenic plants, the coat protein gene must be inserted into the genome of the plant. Furthermore, the coat protein gene must contain all the genetic control sequences necessary for the expression of the gene after it has been incorporated into the plant genome.
Since the coat protein of a potyvirus is produced by the post translation processing of a polyprotein, the coat protein gene isolated from viral RNA does not contain the genetic regulatory sequences needed for gene expression. The coat protein gene does not contain the transcription and translation signals necessary for its expression once transferred and integrated into a plant genome. It must, therefore, be engineered to contain a plant expressible promoter, a translation initiation codon (ATG) and a plant functional poly(A) addition signal (AATAAA) 3' of its translation termination codon.
In the present invention, the nucleotide sequences of the cat protein genes for WMV-II, PRV-p and ZYMV have been determined, and the genes have been inserted into expression vectors to supply them with the necessary genetic regulatory sequences so that the genes can be expressed when incorporated into a plant genome. Plant cells are transformed with the vector construct and the plant cells are induced to regenerate. The resulting plants contain the coat protein genes and produce the coat protein. The production of the protein confers upon the plant an increased resistance to infection by the virus from which the coat protein gene was derived.